Abstract

Aqueous ethanol extracts of powdered stems of Dendrobium loddigesii afforded three new phenolics including three-7-O-ethyl-9-O-(4-hydroxyphenyl)propionyl -diacylglycerol (1), (R)-4,5,4ʹ-trihydroxy-3,3ʹ,α-trimethoxybibenzyl (2) and (S)-5,5′,7-trihydroxy-3′,4′-dimethoxyfavanone (3), together with eleven known analogs. Their structures were determined by extensive spectroscopic analysis. To identify natural antioxidants, whitening, and anti-aging agents, the abilities of these phenolics were assessed to scavenge the 1,2-diphenyl-2-picrylhydrazyl (DPPH) radical, their abilities to inhibit tyrosinase production, and their abilities to stimulate collagen production by human dermal fibroblasts-adult (HDFa) assay. It was found that compounds 1, 4–8, 13, and 14 exhibited significant DPPH radical scavenging activities, compound 10 exhibited tyrosinase inhibitory activity (IC50 37.904 μg/mL), and compound 9 showed significant collagen production with an EC50 value of 3.182 μg/mL. These results suggest that phenolic constituents from D. loddigesii may be candidate antioxidants, skin-whitening, and/or anti-aging agents.

According to relevant studies,cistanche is a common herb that is known as "the miracle herb that prolongs life". Its main component is cistanoside, which has various effects such as antioxidant, anti-inflammatory, and immune function promotion. The mechanism between cistanche and skin whitening lies in the antioxidant effect of cistanche glycosides. Melanin in human skin is produced by the oxidation of tyrosine catalyzed by tyrosinase, and the oxidation reaction requires the participation of oxygen, so the oxygen-free radicals in the body become an important factor affecting melanin production. Cistanche contains cistanoside, which is an antioxidant and can reduce the generation of free radicals in the body, thus inhibiting melanin production.

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Graphic Abstract

1 Introduction

The genus Dendrobium (Orchidaceae) contains approximately 1500 species globally, of which about 80 species grow in China [1]. The stems of several plants in this genus are known as “Shi-Hu”, which have been used for thousands of years as both traditional Chinese medicine and folk remedies for the treatment of chronic atrophic gastritis, skin aging, fever, cardiovascular disease, and a tonic for promoting the production of body fluids [2]. Previous studies on this genus led to the isolation of a series of polysaccharides, phenolic compounds, alkaloids, and sesquiterpenoids [1, 3–5], some of which possess various bioactivities including anti-inflammatory [6], antimicrobial [2], antioxidant [7], antitumor [8], antiplatelet aggregation [9], immunomodulatory [10], and against influenza A activities [11].

Dendrobium loddigesii, a perennial epiphytic herb, is widely distributed in the southwestern area of China, such as Guangxi, Guizhou, and Yunnan Provinces [12]. Its stem has been applied in folk medicine to treat gastritis, fever, and dizziness [13]. In continuing the search for structurally diverse and biologically active natural products from this genus [1, 5, 14–17], an in-depth investigation of pharmacologically active constituents from this plant species was performed herein. As a result, three new phenolic compounds (1–3) as well as eleven known ones (Fig. 1) were isolated from an 80% ethanolic extract of the stem of D. loddigesii. The isolation, structure elucidation, and biological evaluation of these compounds are presented herein.

2 Results and Discussion

Compound 1, obtained as a white solid, gave a molecular formula of C21H26O7 as determined by the (−)-HRESIMS ion at m/z 389.1602 [M−H]− (calcd for C21H25O7, 389.1606) with nine degrees of unsaturation. The 1 H NMR spectrum of 1 contains seven aromatic protons at δH 6.85 (1H, d, J=1.7 Hz), 6.75 (1H, d, J=8.0 Hz), 6.68 (1H, dd, J = 8.0, 1.7 Hz), 7.01 (2H, d, J = 8.5 Hz) and 6.68 (2H, d, J = 8.5 Hz), suggesting the presence of a 1,3,4-trisubstituted benzene ring and a 1,4-disubstituted benzene ring. Its 13C NMR spectrum exhibited 21 carbon resonances including two methyls (one methoxy), four aliphatic methylene, nine methines (two sp3, seven sp2 ), and six quaternary carbons (one carbonyl, five olefinic including three oxygenated). The HMBC correlations (Fig. 2) of H-7/C-1 (δC 131.6), C-2 (δC 111.7), C-6 (δC 121.4), C-8 (δC 74.2), and C-10 (δC 65.3); H-9/C-7 (δC 83.8) and C-8 (δC 74.2); H-10/C-11 (δC 15.6); 3-OMe (δH 3.81)/C-3 (δC 149.1), together with correlations of H-7/H-8/H2-9 and H2-10/H3-11 from 1 H–1 H COSY spectrum (Fig. 2) indicated the presence of 7-O-ethylguaiacylglycerol [18]. It has been reported that J7,8 was about 5 Hz for the erythro isomer and 7 Hz for the three isomers in the cases of syringes-glycerols and diacylglycerol derivatives. Thus, compound 1 was considered to be the three isomers with J7,8 (6.5 Hz) [18]. The HMBC correlations of H-7ʹ (δH 2.79, t, J = 7.5 Hz)/C-8ʹ (δC 37.1), C-1ʹ (δC 132.7), C-2ʹ, 6ʹ (δC 130.2) and C-9ʹ (δC 174.6), H-8ʹ (δH 2.59, t, J = 7.5 Hz)/C-7ʹ (δC 31.0), C-1ʹ, and C-9ʹ, along with COSY cross-peaks of H-8ʹ/H-7ʹ indicated the presence of p-hydroxycoumaric acid [19]. On the basis of the evidence described above, 1 was proposed to have a 7-O-ethylguaiacylglycerol moiety and a p-hydroxy-coumaric acid via an ester linkage. The HMBC correlation from H-9 to C-9ʹ suggested the ester linkage was between C-9 and C-9ʹ. Thus, the structure of 1 was determined as shown.

(R)-4,5,4ʹ-Trihydroxy-3,3ʹ,α-trimethoxybibenzyl (2) was obtained as a white solid. The HRESIMS spectrum of 2 displayed a quasimolecular ion peak at m/z 319.1180 [M−H]− (calcd. for C17H19O6, 319.1187) with 8 degrees of unsaturation. The 1 H NMR spectrum of 2 showed three methoxyl groups at δH 3.75 (3H, s), 3.70 (3H, s), and 3.17 (3H, s); one oxygenated methine proton at δH 4.13 (1H, t, J=6.8 Hz, H-α); two methylene signals at δH 2.73 (1H, dd, J=13.5, 6.9 Hz) and 2.96 (1H, dd, J=13.5, 6.9 Hz); and five aromatic protons, appearing as a 1,3,4,5-tetrasubstituted aromatic ring at δH 6.28 (1H, d, J=1.8 Hz) and 6.34 (1H, d, J=1.8 Hz), and a 1,3,4-trisubstituted aromatic ring at δH 6.49 (1H, d, J=2.0 Hz), 6.62 (1H, d, J=8.0 Hz) and 6.52 (1H, dd, J = 8.0, 2.0 Hz). The 13CNMR and DEPT spectra of 2 showed three oxymethylene, one methylene, one oxygenated methine, and 12 aromatic carbons (five oxygenated). Comparison of its NMR data (Table 1) with those of dendrocandin C [20] showed great similarities except for the presence of one more methoxyl group, which was located at C-3ʹ by the HMBC correlations from 3ʹ-OMe and H-5ʹ to C-3ʹ (δC 148.4). In addition, the multiple HMBC interactions (Fig. 2) of 3-OMe and H-2/C-3 (δC 149.5); α-OMe/C-α (δC 86.9) suggested the other methoxyl groups at C-3, and C-α, respectively. The absolute configuration at C-α was determined as R on the basis of the negative optical rotation ([훼] 26 D –12.46), similar to aphyllous D [훼] 20 D –20.3, MeOH) [15]. Accordingly, the structure of 2 was determined as shown.

(S)-5,7,5′-Trihydroxy-3′,4′-dimethoxyfavanone (3) was obtained as yellow amorphous powder and had the molecular C17H16O7 (10 indices of hydrogen deficiency) according to the (−)-HRESIMS ion at m/z 331.0819 [M – H]− (calcd 331.0823). The UV absorption maxima at 206 and 294 nm indicated the presence of a flavanone [21]. The 1 H NMR spectrum (Table 1) showed three signals in the non-aromatic region placed at δH 5.29 (1H, dd, J=12.7, 3.1, H-2), 3.04 (1H, dd, J=17.1, 12.7, H-3ax), and 2.71 (1H, dd, J=17.1, 3.1, H-3 eq), four aromatic protons δH 5.87 (1H, d, J=2.2, H-6), 5.91 (1H, d, J=2.2, H-8), 6.62 (1H, d, J=2.0, H-2ʹ) and 6.61 (1H, d, J=2.0, H-6ʹ), two methoxyl groups δH 3.83 (3H, s) and 3.77 (3H, s). The 13C NMR and DEPT spectra (Table 1) contain resonances for 17 carbons including two methoxys, one methylene, one methine, one carbonyl carbon, and 12 aromatic carbons. Comprehensive analysis of its NMR data indicated that its planar structure is closely related to that of dihydro tricin [22], except for the OH-4ʹ and OCH3-5ʹ resonances in dihydro tricin were transposed in 3. This was confirmed by the HMBC cross-peaks (Fig. 2) from H-2ʹ, H-6ʹ, and OCH3-4ʹ to C-4ʹ (δC 137.7), from H-6ʹ to C-5ʹ (δC 151.8). The absolute configuration at C-2 was postulated as being in the S-form on the basis of a negative specific rotation value (– 46.64, MeOH) in its optical rotation [23]. Therefore, the structure of compound 3 was thus unambiguously assigned as shown.

Eleven known compounds were identified to be trepidation 4 [24], mescalin 5 [25], 4,5,4′-trihydroxy- 3,3′-dimethoxybibenzyl 6 [26], 4′,5-dihydroxy-3,3′- dimethoxybibenzyl 7 [27], Tristin 8 [28], batatas in III 9 [27], 3,5,3′-hydroxybibenzyl 10 [29], aphyllous C 11 [15], dentiform A 12 [30], dihydroconiferyl dihydro-p-coumarate 13 [31], p-hydroxyphenyl trans-ferulate 14 [32] by spectroscopic analysis and comparing their spectral data with literature.

Phenolic compounds, are an essential part of the human diet and are known as powerful antioxidants due to their potent chain-breaking action and they may contribute directly to the anti-oxidative activity [33]. The DPPH radical scavenging assay is one of the most common and relatively quick methods used for evaluating antioxidant activity. Compounds that can donate a hydrogen atom to the DPPH radical, and then give rise to the reduced form of DPPH will be considered potential antioxidant agents. All compounds were evaluated for their DPPH radical scavenging activities. The present results (Table 2) exhibited that the majority of the phenolic compounds (1, 4–8, 13, and 14) showed significant activities with scavenging capacities ranging from 89.411 to 94.278% at 100 μg/ mL.

On the other hand, tyrosinase is a copper-containing enzyme and plays a critical role in controlling the melanin biosynthesis pathway in melanocytes [34]. Therefore, tyrosinase inhibitors became important constituents of cosmetics or medicinal products for hyperpigmentation and developing skin whitening agents. In the present study, all the isolates were evaluated for their tyrosinase inhibitory activity (Table 2). Kojic acid, a purported skin-lightening agent, was used as a positive control. 3,5,3′-hydroxybibenzyl (10) revealed a significant inhibitory activity with an IC50 value of 37.904 μg/mL. Aphyllals C (11) showed moderate inhibition (IC50, 152.56 μg/mL). All remaining compounds were inactive at concentrations up to 200 μg/mL. In this study, it can be concluded that compounds 10 and 11 can be a potential candidate for the treatment of melanin biosynthesis-related skin diseases.

Considering that this species is medicinally used for skin aging, since, collagen is critical for skin strength and elasticity, and its degradation leads to wrinkles that accompany aging [35]. Hence, all the compounds were also purposely evaluated for their effects on collagen production in HDFa. The results (Table 2) showed that compound 9 significantly stimulation HDFa collagen production activity (EC50 3.182 μg/mL). Compounds 6 and 7 showed weaker activities, with collagen production of 33.062% and 29.157% at 10 μg/mL, respectively. The present results not only supported the ethnopharmacological usage of D. loddigesii but also provided a reliable structure template for developing collagen deficiency-associated diseases such as burns and ulcers.

3 Experimental

3.1 General Experimental Procedures

3.2 Plant Material

The stems of D. loddigesii were collected in September 2014 from Wenshan City, Yunnan Province, People’s Republic of China, and identified by Professor Hong Yu (Yunnan University, Kunming, People′s Republic of China). The voucher specimen (No. 20,140,829) has been deposited at the State Key Laboratory of Phytochemistry and Plant Resource in West China, Kunming Institute of Botany, Chinese Academy of Sciences.

3.3 Extraction and Isolation

The dried and powdered stems (10.2 kg) of D. loddigesii were extracted three times with 80% ethanol under room temperature and concentrated under reduced pressure. Then, the residue was suspended in H2O and partitioned with EtOAc to obtain the EtOAc fraction (220 g), which was subjected to silica gel column chromatography eluted with a gradient of petroleum ether/acetone (15:1 to 0:1) to afford 22 fractions (Fr.1–22). Fr.11 (6 g) was subjected to silica gel CC eluted with CHCl3/MeOH (300:1), then followed by MCI column (MeOH/H2O gradient, 60:40–95:5) and silica gel CC (CHCl3/MeOH, 200:1) to yield 12 (4 mg). Fr.13 (850 mg) was separated over a column of MCI (MeOH/ H2O, 60:40 to 95:5) to give five fractions (Fr.13.1–Fr.13.5). Fr.13.4 (150  mg) afforded compounds 4 (3 mg) and 5 (5 mg) by HPLC preparation (MeOH/H2O, 60:40). Fr.16 (19 g) was chromatographed on silica gel column eluted with CHCl3/MeOH (100:1 to 20:1) to afford 6 subfractions (Fr.16.1–Fr.16.6). Fr.16.4 (2.3 g) was applied MCI column eluted with MeOH/H2O (50:50–100:0) and then further fractionated over a column of Sephadex LH-20 (MeOH/ H2O, 90:10) to yield 7 (716 mg) and 13 (39 mg). By using the same conditions of Fr.16.4, Fr.16.6 (240 mg) afforded compound 9 (7 mg). Fr.18 (10 g) was separated by silica gel CC (CHCl3/MeOH, 100:1–20:1), then passed through MCI (MeOH/H2O gradient, 50:50–100:0) and Sephadex LH-20 (MeOH/H2O, 90:10) columns to yield 3 (25 mg) and 11 (4 mg). Fr.19 (20 g) was subjected to an MCI column eluted with MeOH/H2O (30:70–100:0) to obtain seven fractions (Fr.19.1–Fr.19.7). Fr.19.5 (2.3 g) was separated by repeated silica gel column (CHCl3/MeOH, 30:1) to afford 8 (15 mg) and 10 (7 mg). Fr.19.6 (4.3 g) was fractionated on a column of silica gel (CHCl3/MeOH, 30:1) to give 5 fractions (Fr.19.6.1– Fr.19.6.5). Fr.19.6.1 (1.2 g) was subjected to silica gel column (CHCl3/MeOH, 30:1) and after purification by HPLC (MeOH/H2O, 45:55), providing 1 (15 mg). Fr.19.6.3 (540 mg) was applied to a column of Sephadex LH-20 column eluted with MeOH/H2O (90:10), and then further purified by semipreparative HPLC (MeOH/H2O, 45:55) to furnish 2 (6 mg) and 6 (5 mg). Fr.20 (12 g) was applied to an MCI gel (MeOH/H2O, 30:70–100:0) chromatographic step and then was subjected to silica gel CC (CHCl3/MeOH, 30:1) to give 14 (21 mg). three-7-O-Ethyl-9-O-(4-hydroxyphenyl) propionyl-diacylglycerol (1): white solid; [α]D 26 – 3.72 (c 0.51, MeOH); UV (MeOH) λmax (log ε) 203 (4.53), 225 (4.17), 280 (3.66) nm; IR (KBr) νmax 3426, 1727, 1516, 829 cm−1; 1 H and 13C NMR (CD3OD), see Table 1; ESIMS m/z 389 [M−H]−, HRESIMS m/z 389.1602 [M−H]− (calcd. for C21H25O7, 389.1606).

(R)-4,5,4′-Trihydroxy-3,3ʹ,α-trimethoxybibenzyl (2): white amorphous powder; [α]D 26 –12.46 (c 1.07, MeOH); UV (MeOH) λmax (log ε) 204 (4.59), 286 (3.75) nm; IR (KBr) νmax 3418, 1607, 1517, 1455, 1434, 796 cm−1; 1 H and 13C NMR (CD3OD), see Table 1; ESIMS m/z 319 [M−H]−, HRESIMS m/z 319.1180 [M−H]− (calcd. for C17H19O6, 319.1187).

(2S)-5,7,3ʹ-Trihydroxy-6,4,5-trimethoxyfavone (3): yellow amorphous powder; [α]D 26 – 46.64 (c 0.46, MeOH); UV (MeOH) λmax (log ε) 206 (4.70), 294 (4.17) nm; IR (KBr) νmax 3335, 2940, 1641, 1514, 1462, 1345, 1434, 1182, 1091, 998, 833 cm−1; 1 H and 13C NMR (CD3OD), see Table 1; ESIMS m/z 331 [M−H]−, HRESIMS m/z 331.0819 [M−H]− (calcd. for C17H15O7, 331.0823).

3.4 DPPH Radical Scavenging Activity Assay

The free radical scavenging activity assay was carried out according to the previous method [36] with some modifications. Briefly, 30 μL samples (1000  μg/mL, dissolved in ethanol) and Trolox (1 mM) were added to 270 μL DPPH solution (100 μM, dissolved in methanol), respectively. The reaction proceeded for 1 h at 37 °C on a 96-well microplate. The absorbance was then read at 515 nm and the percentage of total radical scavenging activity was calculated using the following formula: inhibition % =[(A0− A1)/A0]×100%, where A0 is the absorbance of the DPPH without samples (control reaction) and A1 is the absorbance of DPPH incubated with the samples. All the tests were conducted in triplicate and Trolox was used as a positive control agent.

3.5 Mushroom Tyrosinase Inhibitory Assay

Tyrosinase activity inhibition was determined spectrophotometrically according to the method described previously [36] with some modifications. Briefly, different concentrations of test compounds were prepared in 10% DMSO. Each of the sample solution (20 μM) were mixed with L-Dopa (1.25 mM), and diluted with 970 μL of 0.05 mM sodium phosphate buffer (PBS, pH 6.8) in the test tubes. The reaction was initiated by adding mushroom tyrosinase (25 U/mL). The reaction mixture was incubated for 5 min at room temperature. The amount of Dopachrome in the mixture was determined by the measurement of the absorbance of each well at 490 nm. Kojic acid was used as a positive control. The inhibitory percentage of tyrosinase was calculated according to the following equation: Percent inhibition = [(A0− A1)/A0] × 100%, where A0 is the absorbance of the Dopachrome without test compounds (control reaction) and A1 is the absorbance of Dopachrome incubated with the test compounds.

3.6 Collagen Production by HDFa Assay

Acknowledgments This project was supported financially by the Yunnan Provincial Science and Technology Department (Nos. 2017ZF003-04, 2015HB093, and 2019HA001). The authors are grateful to the staff of the analytical group of the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, for measurements of all spectra.

Compliance with Ethical Standards

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References

For more info: david.deng@wecistanche.com  WhatApp:86 13632399501